Polyhydroxyalkanoate production from polyols

ABSTRACT

Organisms are provided which express enzymes such as glycerol dehydratase, diol dehydratase, acyl-CoA transferase, acyl-CoA synthetase β-ketothiolase, acetoacetyl-CoA reductase, PHA synthase, glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase, which are useful for the production of PHAs. In some cases one or more of these genes are native to the host organism and the remainder are provided from transgenes. These organisms produce poly (3-hydroxyalkanoate) homopolymers or co-polymers incorporating 3-hydroxypropionate or 3-hydroxyvalerate monomers wherein the 3-hydroxypropionate and 3-hydroxyvalerate units are derived from the enzyme catalysed conversion of diols. Suitable diols that can be used include 1,2-propanediol, 1,3 propanediol and glycerol. Biochemical pathways for obtaining the glycerol from normal cellular metabolites are also described. The PHA polymers are readily recovered and industrially useful as polymers or as starting materials for a range of chemical intermediates including 1,3-propanediol, 3-hydroxypropionaldehyde, acrylics, malonic acid, esters and amines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/444,031, filedMay 21, 2003, entitled “Polyhydroxyalkanoate Production from Polyols” byFrank A. Skraly and Oliver P. Peoples, which is a continuation of U.S.Ser. No. 09/944,243, filed Aug. 30, 2001, now U.S. Pat. No. 6,576,450,which is a continuation of U.S. Ser. No. 09/366,920, filed Aug. 4, 1999,now U.S. Pat. No. 6,329,183, which claims priority to U.S. provisionalapplication Ser. No. 60/095,329 filed Aug. 4, 1998.

BACKGROUND OF THE INVENTION

This is generally in the field of production of polyhydroxyalkanoates bygenetic engineering of bacterial enzymes.

Numerous microorganisms have the ability to accumulate intracellularreserves of poly [(R)-3-hydroxyalkanoate] (PHA) polymers. PHAs arebiodegradable and biocompatible thermoplastic materials with a broadrange of industrial and biomedical applications (Williams and Peoples,1996, CHEMTECH 26: 38-44). PHAs can be produced using a number ofdifferent fermentation process and recovered using a range of extractiontechniques (reviewed by Braunegg et al. 1998, J. Biotechnol. 65:127-161; Choi and Lee, 1999). Plant crops are also being geneticallyengineered to produce these polymers offering a cost structure in linewith the vegetable oils and direct price competitiveness withpetroleum-based polymers (Williams and Peoples 1996, CHEMTECH 26:38-44;Poirier, Y. 1999, Plant Biotechnology pp. 181-185). PHAs are formed bythe action of a PHA synthase enzyme. As the polymer chains grow, theyform insoluble granules. The PHAs can then be recovered and thenconverted into chemicals or converted into chemicals during the recoveryprocess (Martin et al. PCT WO 97/15681). Therefore, in addition to theirutility as polymers, the PHAs represent a unique mechanism for storingnew chemistries in both microbial and plant crop systems.

PHA copolymers containing 3-hydroxyvalerate (3HV), especially PHBV, havebeen described extensively. Many wild type microorganisms are capable ofproducing 3HV-containing PHAs. PHBV has been produced commercially usingRalstonia eutropha (formerly Alcaligenes eutrophus) from glucose andpropionate and from glucose and isobutyrate (U.S. Pat. No. 4,477,654 toHolmes et al.). A number of other microorganisms and processes are knownto those skilled in the art (Braunegg et al. 1998, Journal ofBiotechnology 65: 127-161). Poly(3HV) homopolymer has been producedusing Chromobacterium violaceum from valerate (Steinbüchel et al., 1993,Appl. Microbiol. Biotechnol. 39:443-449). PHAs containing 3HV units havealso been synthesized using recombinant microorganisms. Escherichia coliharboring the R. eutropha PHA biosynthesis genes has been used toproduce PHBV from glucose and either propionate or valerate (Slater etal., 1992, Appl. Environ. Microbiol. 58:1089-1094) and from glucose andeither valine or threonine (Eschenlauer et al., 1996, Int. J. Biol.Macromol. 19:121-130). Klebsiella oxytoca harboring the R. eutropha PHAbiosynthesis genes has been used to produce PHBV from glucose andpropionate (Zhang et al., 1994, Appl. Environ. Microbiol. 60:1198-1205).R. eutropha harboring the PHA synthase gene of Aeromonas caviae was usedto produce poly(3HV-co-3HB-co-3HHp) from alkanoic acids of odd carbonnumbers (Fukui et al., 1997, Biotechnol. Lett. 19:1093-1097).

PHA copolymers containing 3-hydroxypropionate units have also beendescribed. Holmes et al. (U.S. Pat. No. 4,477,654) used R. eutropha tosynthesize poly(3HP-co-3HB-co-3HV) from glucose and either3-chloropropionate or acrylate. Doi et al. (1990, in E. A. Dawes (ed.),Novel Biodegradable Microbial Polymers, Kluwer Academic Publishers, theNetherlands, pp. 37-48) used R. eutropha to synthesize poly(3HP-co-3HB)from 3-hydroxypropionate, 1,5-pentanediol, 1,7-heptanediol, or1,9-nonanediol. Hiramitsu and Doi (1993, Polymer 34:4782-4786) usedAlcaligenes latus to synthesize poly(3HP-co-3HB) from sucrose and3-hydroxypropionate. Shimamura et al. (1994, Macromolecules 27:4429-4435) used A. latus to synthesize poly(3HP-co-3HB) from3-hydroxypropionate and either 3-hydroxybutyrate or sucrose. The highestlevel of 3-hydroxypropionate incorporated into these copolymers was 88mol % (Shimamura et al., 1994, ibid.). No recombinant 3HP containing PHAproducers have been described in the art.

It is economically desirable to be able to produce these polymers intransgenic crop species. Methods for production of plants have beendescribed in U.S. Pat. No. 5,245,023 and U.S. Pat. No. 5,250,430; U.S.Pat. No. 5,502,273; U.S. Pat. No. 5,534,432; U.S. Pat. No. 5,602,321;U.S. Pat. No. 5,610,041; U.S. Pat. No. 5,650,555: U.S. Pat. No.5,663,063; WO 9100917, WO 9219747, WO 9302187, WO 9302194 and WO9412014, Poirier et. al., 1992, Science 256; 520-523, Williams andPeoples, 1996. Chemtech 26, 38-44, the teachings of which areincorporated by reference herein). In order to achieve this goal, it isnecessary to transfer a gene, or genes in the case of a PHA polymerasewith more than one subunit, encoding a PHA polymerase from amicroorganism into plant cells and obtain the appropriate level ofproduction of the PHA polymerase enzyme. In addition it may be necessaryto provide additional PHA biosynthetic genes, e.g. a ketoacyl-CoAthiolase, an acetoacetyl-CoA reductase gene, a 4-hydroxybutyryl-CoAtransferase gene or other genes encoding enzymes required to synthesizethe substrates for the PHA polymerase enzymes. In many cases, it isdesirable to control the expression in different plant tissues ororganelles. Methods for controlling expression in plant tissues ororganelles are known to those skilled in the art (Gasser and Fraley,1989, Science 244; 1293-1299; Gene Transfer to Plants, 1995, Potrykus,I. and Spangenberg, G. eds. Springer-Verlag Berlin Heidelberg New York.and “Transgenic Plants: A Production System for Industrial andPharmaceutical Proteins”, 1996, Owen, M. R. L. and Pen, J. Eds. JohnWiley & Sons Ltd. England, incorporated herein by reference).

Although methods for production of a variety of different copolymers inbacterial fermentation systems are known, and production of PHAs inplants has been achieved, the range of copolymers possible in bacteriahas not been achieved in plants. It would be advantageous to be able toproduce different copolymers in transgenic plants, and to have moreoptions with regard to the substrates to be utilized by the transgenicplants.

It is therefore an object of the present invention to provide methodsand reagents for production of PHAs in plants.

It is a further object of the present invention to provide methods andreagents for production of PHAs using simple sugars and alcohols assubstrates.

It is still another object of the present invention to provide methodsand reagents for production of copolymers other than PHB and PHVB.

SUMMARY OF THE INVENTION

Organisms are provided which express enzymes such as glyceroldehydratase, diol dehydratase, acyl-CoA transferase, acyl-CoA synthetaseβ-ketothiolase, acetoacetyl-CoA reductase, PHA synthase,glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase, which areuseful for the production of PHAs. In some cases one or more of thesegenes are native to the host organism and the remainder are providedfrom transgenes. These organisms produce poly (3-hydroxyalkanoate)homopolymers or co-polymers incorporating 3-hydroxypropionate or3-hydroxyvalerate monomers wherein the 3-hydroxypropionate and3-hydroxyvalerate units are derived from the enzyme catalysed conversionof diols. Suitable diols that can be used include 1,2-propanediol, 1,3propanediol and glycerol. Biochemical pathways for obtaining theglycerol from normal cellular metabolites are also described. The PHApolymers are readily recovered and industrially useful as polymers or asstarting materials for a range of chemical intermediates including1,3-propanediol, 3-hydroxypropionaldehyde, acrylics, malonic acid,esters and amines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of the production of 3-hydroxyvaleryl-CoA fromglycerol-3-P.

FIG. 2 is a schematic of the plasmid construct pFS44C encoding glyceroldehydratase (dhaB) and 4-hydroxybutyryl-CoA transferase (hbcT).

FIG. 3 is a schematic of the plasmid construct pFS45 encoding dhaB, hbcTand phaC.

FIG. 4 is a schematic of the plasmid construct pFS47A, encoding dhaT,dhaB, and hbcT.

FIG. 5 is a schematic of the plasmid construct pFS48B, encoding dhaT,dhaB, hbcT, and phaC.

FIG. 6 is a schematic of the plasmid construct pMS15, encoding dhaT,DAR1-GPP2 (DAR1, dihydroxyacetone phosphate dehydrogenase; and GPP2,sn-glycerol-3-phosphate phosphatase), dhaB, hbcT, and phaC.

FIG. 7 is a schematic of the plasmid construct pFS51, encoding GPP2 andDAR1.

DETAILED DESCRIPTION OF THE INVENTION

New metabolic pathways have been developed for the production of PHAscontaining 3-hydroxyvalerate units from 1,2-propanediol and of PHAscontaining 3-hydroxypropionate units from 1,3 propanediol or glycerol.In the case of glycerol, the glycerol can either be fed to themicroorganism or can be produced from central metabolic intermediates.The key enzymes components of these novel metabolic pathways leading tothese monomers and their polymerization are illustrated in FIG. 1.

1,2-propanediol and glycerol are inexpensive substrates that are nontoxic to many microorganisms even at high concentrations.1,3-propanediol can be produced from renewable resources (Anton, D.“Biological production of 1,3-propanediol”, presented at UnitedEngineering Foundation Metabolic Engineering II conference, Elmau,Germany, Oct. 27, 1998). 1,2-propanediol is present in industrial wastestreams from production of propylene glycol. Glycerol can also beobtained from metabolism in a number of microbes and plant crops. Inmany cases, these are superior feedstocks for fermentation as comparedto organic acids, which generally become toxic at low concentrations tomany microorganisms. 3-Hydroxypropionic acid is not chemically stableand therefore is not commercially available.

Organisms to be Engineered

In one embodiment, genes for the entire pathway illustrated in FIG. 1are introduced into the production organism. An organism that does notnaturally produce PHAs, such as Escherichia coli, may be used. A numberof recombinant E. coli PHB production systems have been described(Madison and Huisman, 1999, Microbiology and Molecular Biology Reviews,63: 21-53). The genes encoding a vicinal diol dehydratase, from anorganism that naturally can convert glycerol to 3-hydroxypropionaldehyde(Klebsiella pneumoniae, e.g.), are introduced into this host. In thecase of 1,2-propanediol, the vicinal diol dehydratase converts thesubstrate to propionaldehyde, which can be converted to propionyl-CoA bythe endogenous metabolism of the microorganism, optionally with the aidof an exogenous acyl-CoA transferase or acyl-CoA synthetase. It may beuseful to mutagenize and select strains with increased resistance topropionaldehyde. Propionyl-CoA can then be accepted by the ketoacyl-CoAthiolase in a condensation with acetyl-CoA, thus forming3-hydroxyvaleryl-CoA. The ketoacyl-CoA thiolase will also condenseacetyl-CoA with acetyl-CoA, thus forming 3-hydroxybutyryl-CoA. Both3-hydroxyvaleryl-CoA and 3-hydroxybutyryl-CoA can be accepted by variousPHA synthases such as the one expressed in the recombinant host, andtherefore PHBV is synthesized by the recombinant host.

The host described above can also be fed 1,3 propanediol or glyceroleither during growth or after a separate growth phase, and a 3HP polymeris accumulated within the cells. E. coli does not synthesize coenzymeB-12 de novo, and therefore coenzyme B-12 or a precursor that E. colican convert to coenzyme B-12, such as vitamin B-12, must also be fed. Inthe case of glycerol, the vicinal diol dehydratase converts thesubstrate to 3-hydroxypropionaldehyde, which can be converted to3-hydroxypropionyl-CoA by the endogenous metabolism of themicroorganism, optionally with the aid of an exogenous acyl-CoAtransferase or acyl-CoA synthetase. 3-Hydroxypropionyl-CoA may then bepolymerized by PHA synthase to P3HP. Hydroxyacyl-CoA monomer units inaddition to 3HP may also be incorporated into the polymer. Ifketoacyl-CoA thiolase and reductase are expressed, for example, then acopolymer of 3-hydroxybutyrate and 3-hydroxypropionate can be formed.

In order to produce the 3HP polymers directly from carbohydratefeedstocks, the E. coli is further engineered to expressglycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Suchrecombinant E. coli strains and methods for their construction are knownin the art (Anton, D. “Biological production of 1,3-propanediol”,presented at United Engineering Foundation Metabolic Engineering IIconference, Elmau, Germany, Oct. 27, 1998; PCT WO 98/21339).

In another embodiment, a recombinant organism that naturally contains avicinal diol dehydratase can be used. One example of such an organism isKlebsiella oxytoca, although several others exist, as discussed above.In this embodiment no exogenous vicinal diol dehydratase need beimported from another organism. However it may be useful to mutagenisethis organism and select mutants that express the dehydratase duringaerobic growth or it can be genetically engineered to express the geneunder aerobic conditions. It is generally the case that organisms whichcontain one or more coenzyme B-12-dependent vicinal diol dehydratasescan synthesize coenzyme B-12 de novo, and in those cases it is notnecessary to add coenzyme B-12 or closely related precursors thereof toany part of the cultivation. In this case, a PHA synthase or an entirePHB biosynthetic pathway and optionally an exogenous acyl-CoAtransferase or acyl-CoA synthetase is introduced into this organism.Techniques for doing this are well known in the art (for example, Denniset al., 1998, Journal of Biotechnology 64:177-186). In order to producethe 3HP polymers directly from carbohydrate feedstocks, the strain isfurther engineered to express glycerol-3-phosphate dehydrogenase andglycerol-3-phosphatase as described above.

In another embodiment, an organism that naturally produces PHAs can beused. Examples of such organisms include Ralstonia eutropha, Alcaligeneslatus and Azotobacter but many others are well-known to those skilled inthe art (Braunegg et al. 1998, Journal of Biotechnology 65: 127-161).The introduction of the diol dehydratase is accomplished using standardtechniques as described by Peoples and Sinskey (1989, J. Biol. Chem.164, 15298-15303). In these cases it may be useful to mutate theorganism and select for increased resistance to3-hydroxypropionaldehyde. PHA-producing organisms vary in their abilityto synthesize coenzyme B-12 de novo, and therefore coenzyme B-12 or aprecursor which the organism can convert to coenzyme B-12 would be addedas appropriate. PHBV is then produced by feeding 1,2 propanediol and atleast one other feedstock. PHBP is produced by feeding 1,3 propanediolor glycerol and one other feedstock, for example, glucose. In order toproduce the 3HP polymers directly from carbohydrate feedstocks, thestrain is further engineered to express glycerol-3-phosphatedehydrogenase and glycerol-3-phosphatase as described above. It may beuseful to utilize mutations that are beneficial for the production ofthe P3HP homopolymers in these organisms. Specific mutations includeinactivating the β-ketothiolase and/or acetoacetyl-CoA reductase genes.As these genes are generally well known and available or isolatable,gene disruptions can be readily carried out as described for example bySlater et. al., 1998 (J. Bacteriol.) 180(8):1979-87.

The implementation of the production of poly(3-hydroxypropionate) andits copolymers is also not limited to bacteria as described in theexamples. The same genes may be introduced into eukaryotic cells,including but not restricted to, yeast and plants, which, like bacteria,also produce glycolytic intermediates such as dihydroxyacetonephosphate, from which glycerol and ultimately poly(3-hydroxypropionate)may be derived.

Genes for Utilization of Substrates

Genes and techniques for developing recombinant PHA producers suitablefor use as described herein are generally known to those skilled in theart (Madison and Huisman, 1999, Microbiology and Molecular BiologyReviews, 63: 21-53; PCT WO 99/14313, which are incorporated herein byreference). Because all of the genes necessary to implement theproduction of poly(3-hydroxypropionate) from central metabolicintermediates via glycerol have been cloned and are available ingenetically manipulatable form, any combination of plasmid-borne andintegrated genes may be used, and the implementation of this pathway istherefore not restricted to the schemes outlined herein. Many differentimplementations will be apparent to those skilled in the art.

Glycerol dehydratase (EC 4.2.1.30) and diol dehydratase (EC 4.2.1.28)are distinct coenzyme B-12-requiring enzymes found in several species ofbacteria. Often glycerol dehydratase is induced during anaerobic growthon glycerol and diol dehydratase is induced during anaerobic growth oneither glycerol or 1,2-propanediol (Forage and Foster, 1979, Biochim.Biophys. Acta 569:249-258). These dehydratases catalyze the formation of3-hydroxypropionaldehyde from glycerol and propionaldehyde from1,2-propanediol. These aldehydes are usually converted to thecorresponding alcohols by a dehydrogenase. Organisms that contain one orboth dehydratases typically are able to convert glycerol to3-hydroxypropionaldehyde or 1,3-propanediol. Bacterial species noted forthis ability include Klebsiella pneumoniae (Streekstra et al., 1987,Arch. Microbiol. 147: 268-275), Klebsiella oxytoca (Homann et al., 1990,Appl. Microbiol. Biotechnol. 33: 121-126), Klebsiella planticola (Homannet al., 1990, ibid.) and Citrobacter freundii (Boenigk et al., 1993,Appl. Microbiol. Biotechnol. 38: 453-457) although many other examplesare generally known. Both dehydratases are formed of three subunits,each of which is homologous to its counterpart in the other enzyme.

The substrate range of the glycerol and diol dehydratases (which willalso be referred to generically from this point on as “vicinal dioldehydratases”) is not limited to glycerol and 1,2-propanediol.Bachovchin et al. (1977, Biochemistry 16:1082-1092), for example,demonstrated that the substrates accepted by the K. pneumoniae enzymeinclude glycerol, (R)-1,2-propanediol, (S)-1,2-propanediol, ethyleneglycol, thioglycerol, 3-chloro-1,2-propanediol, 1,2-butanediol,2,3-butanediol, isobutylene glycol, and 3,3,3-trifluoro-1,2-propanediol.In all cases, the product of the reaction is the aldehyde or ketoneformed by the effective removal of a water molecule from the substrate.

Organisms that naturally produce glycerol from sugars throughphosphoglycerate include Bacillus licheniformis (Neish et al., 1945,Can. J. Res. 23B: 290-296), Lactobacillus sp. (Nelson and Werkman, 1935,J. Bacteriol. 30: 547-557), Halobacterium cutirubrum (Wassef et al.,1970, Can. J. Biochem. 48: 63-67), Microcoleus chthonoplastes (Moezelaaret al., 1996, Appl. Environ. Microbiol. 62: 1752-1758), Zymomonasmobilis (Viikari, 1988, CRC Crit. Rev. Biotechnol. 7:237-261),Phycomyces blakesleeanus (Van Schaftiger. and Van Laere, 1985, Eur. J.Biochem. 148: 399-405), Saccharomyces cerevisiae (Tsuboi and Hudson,1956, Arch. Biochem. Biophys. 61: 197-210), Saccharomyces carlsbergensis(Tonino and Steyn-Parvë, 1963, Biochim. Biophys. Acta 67: 453-469),Rhizopus javanicus (Lu et al., 1995, Appl. Biochem. Biotechnol. 51/52:83-95), Candida magnoliae (Sahoo, D. K., 1991, Ph.D. Thesis, IndianInstitute of Technology, Delhi), Candida utilis (Gancedo et al., 1968,Eur. J. Biochem. 5: 165-172), Aspergillus niger (Legisa and Mattey,1986, Enzyme Microb. Technol. 8: 607-609), Trichomonas vaginaiis(Steinbüchel and Müller, 1986, Molec. Biochem. Parasitol. 20: 45-55),Dunaliella salina (Sussman and Avron, 1981, Biochim. Biophys. Acta 661:199-204; Ben-Amotz et al., 1982, Experientia 38: 49-52), Asteromonasgracilis (Ben-Amotz et al., ibid.), Leishmania mexicana (Cazzulo et al.,1988, FEMS Microbiol. Lett. 51: 187-192), and Crithidia fasciculata(Cazzulo et al., ibid.). In many of these organisms, glycerol is knownto be derived from dihydroxyacetone phosphate, an intermediate of theglycolytic pathway. Escherichia coli does not normally synthesizeglycerol in significant amounts when grown on most sugars (Baldomà andAguilar, J. Bacteriol. 170:416, 1988). However, transgenic E. colistrains that can form glycerol from common sugars such as glucose havebeen described, for example, in PCT WO 97/20292.

Genetically engineered systems for the production of glycerol fromsugars (WO 98/21339), the production of 1,3-propanediol from glycerol(WO 96/35796, WO 98/21339) and the production of 1,3-propanediol fromsugars have been described. E. coli expressing the DAR1(dihydroxyacetone phosphate dehydrogenase) and GPP2(sn-glycerol-3-phosphate phosphatase) genes of Saccharomyces cerevisiaewere shown to accumulate high concentrations of glycerol in the mediumwhen grown on glucose (Anton, D. “Biological production of1,3-propanediol”, presented at United Engineering Foundation MetabolicEngineering II conference, Elmau, Germany, Oct. 27, 1998).

Regulation of Expression

In any of the aforementioned embodiments, it is possible to control thecomposition of the polymer produced by controlling the expression of thevicinal diol dehydratase or by controlling the availability of coenzymeB-12. The higher the dehydratase activity, the more activated monomerwill be derived as a result of its activity, up to the point whereanother factor such as substrate availability or an enzyme activitydownstream of the dehydratase becomes limiting. Methods for modulationof gene expression (and thus enzyme activity) in various organisms arewell-known to those skilled in the art. An additional method for thecontrol of vicinal diol dehydratase activity is the modulation of theavailability of coenzyme B-12 to the microorganism. Many strains ofEscherichia coli, for example, are unable to synthesize coenzyme B-12 denovo, and therefore recombinant vicinal diol dehydratase, which dependsupon coenzyme B-12 for activity, is not active in these strains unlesscoenzyme B-12 or a suitable precursor such as vitamin B-12 is added tothe medium. In Escherichia coli strains which harbor PHA synthesis genesand a vicinal diol dehydratase, it has been found that with no coenzymeB-12 addition, only PHB is synthesized even though 1,2-propanediol ispresent in the medium. The addition of 1 μM coenzyme B-12 to acultivation of the same strain in the same medium leads to PHBVformation as discussed in the examples. Skraly et al. (1998, Appl.Environ. Microbiol. 64:98-105) found that transgenic Escherichia colisynthesized increasing levels of 1,3-propanediol from glycerol asincreasing concentrations of coenzyme B-12 were provided in the medium,up to a concentration of about 20 nM, after which the 1,3-propanediolyield did not increase. Therefore, coenzyme B-12 concentrations from 0to 20 nM can be used to control the PHBV composition in Escherichia coliharboring PHA synthesis genes and a vicinal diol dehydratase genecultivated in a medium containing 1,2-propanediol. The same basicpremise is true for deriving poly(3-hydroxypropionate) from glycerol.The cells are able to make a PHA (such as PHB) in the presence ofcomonomer when no vicinal diol dehydratase is present. The use ofcoenzyme B-12 to control polymer composition can be accomplished withany microorganism that is unable to synthesize coenzyme B-12 de novo.Such organisms include those that naturally lack this ability (such asEscherichia coli) and those that naturally possess this ability (such asKlebsiella pneumoniae) but have been mutated by the use of chemicalmutagenesis or by genetic methods such as transposon mutagenesis to losethis ability.

In the case of some microorganisms, some of the genes can be integratedinto the host chromosome and others provided on a plasmid. In somecases, compatible plasmid systems can be used, for example, with severalsteps on the pathway encoded on one plasmid and the other steps encodedby a second plasmid. A combination of the two approaches may also beused.

Substrates

As discussed above, substrates that can be used to make PHAs includeglycerol and glucose. A number of other substrates, in addition toglycerol or glucose, can be used successfully. Examples of othersubstrates include starch, sucrose, lactose, fructose, xylose,galactose, corn oil, soybean oil, tallow, tall oil, fatty acids orcombinations thereof.

The present invention will be further understood by reference to thefollowing non-limiting examples.

Example 1 PHBV Production from Glucose and 1,2-propanediol

Escherichia coli strain MBX769 (Huisman et. al. PCT WO 99/14313), whichexpresses the PHA synthesis genes from Zoogloea ramigera(acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHA synthase)containing plasmid pFS44C was used to synthesize PHBV from glucose and1,2-propanediol. Plasmid pFS44C (shown schematically in FIG. 2) containsthe genes encoding Klebsiella pneumoniae glycerol dehydratase (dhaB),isolated from pTC53 (Skraly et al., 1998, Appl. Environ. Microbiol.64:98-105), and the Clostridium kluyveri 4-hydroxybutyryl-CoAtransferase (hbcT), isolated from pCK3 (Söhling and Gottschalk, 1996, J.Bacteriol. 178:871-880), both in one operon under control of the trcpromoter. pFS44C also contains the lac repressor gene (lacI), anampicillin resistance gene (ampR), and an origin of replication (ORI),all derived from the vector pSE380) (Invitrogen; La Jolla, Calif.).

The cells were precultured in 100 mL of a medium containing 25 g/L of LBbroth powder (Difco; Detroit, Mich.) and 100 mg/L ampicillin. They wereremoved from this medium by centrifugation (2000×g, 10 minutes) andresuspended in 100 mL of a medium containing, per liter: 5 g LB brothpowder; 50 mmol potassium phosphate, pH 7; 10 g 1,2-propanediol; 2 gglucose; 1 μmol coenzyme B-12; 100 μg ampicillin; and 0.1 mmolisopropyl-β-D-thiogalactopyranoside (IPTG). The cells were incubated inthis medium with shaking at 225 rpm at 30° C. for 48 hours. They werethen removed by centrifugation as above, washed once with water, andlyophilized.

The same experiment was done in parallel, except that no coenzyme B-12was added. About 25 mg of lyophilized cell mass from each flask wassubjected to simultaneous extraction and butanolysis at 110° C. for 3hours in 2 mL of a mixture containing (by volume) 90% 1-butanol and 10%concentrated hydrochloric acid, with 2 mg/mL benzoic acid added as aninternal standard. The water-soluble components of the resulting mixturewere removed by extraction with 3 mL water. The organic phase (1 μL at asplit ratio of 1:50 at an overall flow rate of 2 mL/min) was analyzed onan SPB-1 fused silica capillary GC column (30 m; 0.32 mm ID; 0.25 μmfilm; Supelco; Bellefonte, Pa.) with the following temperature profile:80° C., 2 min; 10 C.° per min to 250° C.; 250° C., 2 min. The standardused to test for the presence of 3-hydroxybutyrate and 3-hydroxyvalerateunits in the polymer was PHBV (Aldrich Chemical Co.; Milwaukee, Wis.).The polymer in the experiment with coenzyme B-12 added accounted for60.9% of the dry cell weight, and it was composed of 97.4%3-hydroxybutyrate units and 2.6% 3-hydroxyvalerate units.

The supernatant at the conclusion of this experiment was found byhigh-performance liquid chromatographic (HPLC) analysis to contain 0.41g/L propanol, indicating that the glycerol dehydratase was functional.The polymer in the experiment with no coenzyme B-12 added accounted for56.7% of the dry cell weight, and it was PHB homopolymer. Thesupernatant at the conclusion of this experiment did not containpropanol. HPLC analysis was done with an Aminex HPX-87H column withsulfuric acid (pH 2) as the mobile phase at a flow rate of 0.6 mL/minand a column temperature of 50° C. Detection was by both refractiveindex and ultraviolet absorption.

Example 2 PHBV and Growth from 1,2-propanediol as Sole Carbon Source

MBX 184 was selected for growth on 1,2-PD, to yield E. coli strain MBX1327. MBX1327 was transduced with the PHB genes ABC5KAN from MBX1164 toyield E. coli strain MBX 1329. MBX1164 is MBX247::ABC5KAN (encoding thethiolase, reductase and PHB synthase from Z ramigera). MBX247 is LJ5218(Spratt, et al. 1981 J. Bacteriol. 146:1166-1169) E. coli genetic stockcenter CGSC 6966) mutagenized with 1-methyl-3-nitro-1-nitrosoguanidine(NTG) by a standard procedure (Miller, J., A short course in bacterialgenetics, 1992, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.), and screened for the ability to grow with 1,2-propanediol as solecarbon source. Strains of E. coli with this ability and methods forgeneration of such strains have been described previously (Sridhara etal., 1969, J. Bacteriol. 93:87). E. coli strain MBX1329 has both thecapability to grow with 1,2-propanediol as the sole carbon source and tosynthesize PHB from carbon sources that generate acetyl-CoA.

MBX1329 harboring plasmid pFS44C (shown in FIG. 2) was grown in a mediumcontaining, per liter: 6.25 g LB broth powder; 3.5 g sodium ammoniumphosphate; 7.5 g dibasic potassium phosphate trihydrate; 3.7 g monobasicpotassium phosphate; 0.12 g magnesium sulfate; 2.78 mg iron (II) sulfateheptahydrate; 1.98 mg manganese (II) chloride tetrahydrate; 2.81 mgcobalt (II) sulfate heptahydrate; 1.47 mg calcium chloride dihydrate;0.17 mg coppe; (II) chloride dihydrate; 0.29 mg zinc (II) chlorideheptahydrate; 10 mg thiamine; 10 g 1,2-propanediol; 50 mol coenzymeB-12; 100 μg ampicillin; and 0.05 mmolisopropyl-β-D-thiogalactopyranoside (IPTG). The total volume was 50 mLin a 250-mL Erlenmeyer flask; the inoculum was 0.5 mL of an overnightculture in 25 g/L LB broth powder and 100 μg/mL ampicillin. The cellswere incubated in this medium for 3 days at 37° C. with shaking at 200rpm. They were removed from this medium by centrifugation (2000×g, 10minutes), washed once with water, centrifuged again, and lyophilized.

Intracellular polymer content was analyzed by butanolysis as inExample 1. The cells grew to an optical density (at 600 nm) of 9.8 andcontained PHBV to 6% of the dry cell weight. The polymer itself wascomposed of 2.5% 3-hydroxyvalerate units and 97.5% 3-hydroxybutyrateunits.

Example 3 Poly(3-hydroxypropionate) from 1,3-propanediol and1,3-propanediol from glycerol

Escherichia coli strain MBX184, which is deficient in the fadR gene andexpresses the atoC gene constitutively, was used to synthesize1,3-propanediol from glycerol and poly(3-hydroxypropionate) from1,3-propanediol. In both instances the cells harbored plasmid pFS45(shown schematically in FIG. 3) which contains genes encoding Klebsiellapneumoniae glycerol dehydratase, Clostridium kluyveri4-hydroxybutyryl-CoA transferase, and Ralstonia eutropha PHA synthase,all in one operon under the control of the trc promoter. The cells werecultivated as described in Example 1, except that glycerol was presentinstead of 1,2-propanediol.

HPLC analysis showed that the cells in the coenzyme-B12-containingmedium synthesized 1.3 g/L of 1,3-propanediol while the cells in themedium free of coenzyme B-12 did not synthesize any 1,3-propanediol. Thesame strain was also cultivated using the method of Example 1 exceptthat 1,3-propanediol was present instead of 1,2-propanediol, and nocoenzyme B-12 was added.

Lyophilized cell mass was analyzed by GC as in Example 1, with anadditional standard of beta-propiolactone to quantifypoly(3-hydroxypropionate). These cells were shown by GC analysis tocontain poly(3-hydroxypropionate) homopolymer at 7.8% of the dry cellweight. The synthesis of poly(3-hydroxypropionate) from glycerol likelydid not occur because of the accumulation of 3-hydroxypropionaldehyde,which is very toxic to many microorganisms (Dobrogosz et al., 1989,Wenner-Gren Int. Symp. Ser. 52:283-292). This toxicity may be addressedby discouraging the accumulation of 3-hydroxypropionaldehyde in at leasttwo ways: 1) a 1,3-propanediol oxidoreductase from a1,3-propanediol-producing organism such as those mentioned above canalso be expressed, and 2) the activity of the unidentified endogenousdehydrogenase from Escherichia coli that is responsible for the observedformation of 1,3-propanediol when glycerol dehydratase is present can beincreased by screening for Escherichia coli cells expressing glyceroldehydratase that grow well in the presence of both glycerol and coenzymeB-12. The second approach can be accomplished, for example, bytransforming mutagenized Escherichia coli with a plasmid such as pFS45,so that the mutagenesis does not affect the glycerol dehydratase gene,followed by enrichment in a medium containing glycerol and coenzymeB-12.

Example 4 Poly(3-hydroxypropionate) from Glycerol

The two pathways in Example 3 (glycerol to 1,3-propanediol and1,3-propanediol to poly(3-hydroxypropionate) were activated in the samerecombinant Escherichia coli. E. coli strain MBX820, which stablyexpresses the PHA biosynthetic genes phaA, phaB, and phaC from Zoogloearamigera, was transformed with the plasmid pFS47A (shown schematicallyin FIG. 4), which contains, under control of the trc promoter, the genesencoding 4-hydroxybutyryl-CoA transferase from Clostridium kluyveri andglycerol dehydratase and 1,3-propanediol oxidoreductase from Klebsiellapneumoniae. PFS47A was constructed from the plasmid pFS16, a predecessorof pFS47A, as follows: The Clostridium kluyveri orfZ gene was amplifiedby PCR from plasmid pCK3 (Söhling and Gottschalk, 1996, J. Bacteriol.178: 871-880) using the following oligonucleotide primers:

(SEQ ID NO: 1) 5′- TCCCCTAGGATTCAGGAGGTTTTTATGGAGTGGGAAGAGATATATAAAG- 3′(orfZ 5′ AvrII) (SEQ ID NO: 2)5′-CCTTAAGTCGACAAATTCTAAAATCTCTTTTTAAATTC-3′ (orfZ 3′ SalI)The orfZ PCR product was ligated to pTrcN, which had been digested withXbaI (which is compatible with AvrII) and SalI.

The cells were precultured in 100 mL of a medium containing 25 g/L of LBbroth powder (Difco; Detroit, Mich.) and 100 mg/L ampicillin. They wereremoved from this medium by centrifugation (2000×g, 10 minutes) andresuspended in 100 mL of a medium containing, per liter: 2.5 g LB brothpowder; 50 mmol potassium phosphate, pH 7; 5 g substrate (glycerol;1,2-propanediol; or 1,3-propanediol); 2 g glucose; 5 nmol coenzyme B-12;100 μg ampicillin; and 0.1 mmol isopropyl-β-D-thiogalactopyranoside(IPTG). The cells were incubated in this medium with shaking at 225 rpmat 30° C. for 48 hours. They were then removed by centrifugation asabove, washed once with water, and lyophilized.

The lyophilized cell mass was analyzed by GC analysis as in Example 1,with an additional standard of beta-propiolactone to quantifypoly(3-hydroxypropionate). The cells cultivated in glycerol and1,3-propanediol both contained a copolymer of 3-hydroxybutyrate and3-hydroxypropionate units, and the cells cultivated in 1,2-propanediolcontained a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate units.Polymer compositions and quantities as a percentage of dry cell weightare given in Table 1. The glycerol-cultivated cells synthesized morepolymer than the 1,3-propanediol-cultivated cells, but the percentage of3-hydroxypropionate units was smaller in the glycerol-cultivated cells.These differences may be explained by the fact that3-hydroxypropionaldehyde is toxic and that it is probably generated morequickly by 1,3-propanediol oxidoreductase from 1,3-propanediol than itis by glycerol dehydratase from glycerol. The toxicity of3-hydroxypropionaldehyde can negatively impact cell health and thereforeoverall polymer content, but its formation from glycerol is necessaryfor 3-hydroxypropionyl-CoA formation whether the necessary intermediateis 3-hydroxypropionaldehyde or 1,3-propanediol.

TABLE 1 Polymers produced by MBX820/pFS47A cultivated in varioussubstrates. 3HB units 3HP units 3HV units Total polymer (% of (% of (%of Substrate (% of dcw^(a)) polymer) polymer) polymer) glycerol 55.898.2 1.8 0.0 1,2-propanediol 41.3 97.1 0.0 2.9 1,3-propanediol 26.7 95.14.9 0.0 ^(a)percent of dry cell weight.

Example 5 Control of Polymer Composition by Variation of Coenzyme B-12Concentration

Because the vicinal diol dehydratases depend upon coenzyme B-12 foractivity, and because the formation of 3-hydroxypropionyl-CoA fromglycerol or of propionyl-CoA from 1,2-propanediol depends upondehydratase activity, the composition of the copolymer in either casecan be controlled by variation of the availability of coenzyme B-12 tothe dehydratase. In this example, this was accomplished by variation ofcoenzyme B-12 concentration added to the medium in which E. coli strainMBX820 carrying the plasmid pFS47A was producing PHA.

The cells were precultured in 100 mL of a medium containing 25 g/L of LBbroth powder (Difco; Detroit, Mich.) and 100 mg/L ampicillin. They wereremoved from this medium by centrifugation (2000×g, 10 minutes) andresuspended in 100 mL of a medium containing, per liter: 2.5 g LB brothpowder; 50 mmol potassium phosphate, pH 7; 10 g substrate (glycerol or1,2-propanediol); 2 g glucose; 100 μg ampicillin; 0.1 mmolisopropyl-β-D-thiogalactopyranoside (IPTG); and 0, 5, 20, or 50-nmolcoenzyme B-12. The cells were incubated in this medium with shaking at225 rpm at 30° C. for 72 hours. They were then removed by centrifugationas above, washed once with water, and lyophilized. The lyophilized cellmass was analyzed by GC analysis as in Example 4.

Table 2 shows the amounts and compositions of the PHAs produced in thisway. The absence of coenzyme B-12, whether the substrate was glycerol or1,2-propanediol, resulted in synthesis of only PHB. Glycerol was moreconducive to PHA formation in the absence of dehydratase activity, asshown by the final optical density and polymer content, presumablybecause E. coli can utilize glycerol as a carbon and energy source underaerobic conditions (Lin, Ann. Rev. Microbiol. 30:535, 1976), whilegenerally this is not true of 1,2-propanediol (Baldomà and Aguilar,ibid.). When coenzyme B-12 is added in increasing amounts to cellscultivated with glycerol, the percentage of 3-hydroxypropionate units inthe polymer increases, while the overall polymer content decreases. Thisdecrease is probably due to the toxicity of 3-hydroxypropionaldehyde,which results in decreased health of the cells. When coenzyme B-12 isadded in increasing amounts to cells cultivated with 1,2-propanediol,3-hydroxyvalerate units are incorporated into the polymer, but thepercentage of 3-hydroxyvalerate in the polymer does not increase to thesame extent as the percentage of 3-hydroxypropionate units did in theglycerol experiment. This indicates that the concentration of coenzymeB-12 is not limiting to 3-hydroxyvaleryl-CoA synthesis when itsconcentration reaches even a few nanomolar, and that some other factorbecomes limiting.

This example demonstrates that the composition of PHAs derived from theuse of coenzyme B-12 dependent dehydratases can be controlled by varyingthe concentration of coenzyme B-12 made available to the dehydratase.The extent to which the control can be executed is dependent on the diolsubstrate used. This can be due to the preference of the vicinal diolfor certain substrates over others and on the rest of the hostmetabolism leading from the aldehyde derived from the diol to theacyl-CoA which serves as the activated monomer for PHA formation.

TABLE 2 Composition of polymers produced by MBX820/pFS47A from glyceroland 1,2-propanediol in media with various coenzyme B-12 concentrations.[CoB12], OD^(a) 3HB^(b), 3HV^(c), 3HP^(d), polymer, Substrate nM (600nm) % of PHA % of PHA % of PHA % of dcw^(e) glycerol 0 19.3 100 0 0 65.45 17.7 81.4 0 18.6 56.6 20 10.0 79.6 0 20.4 45.9 50 3.9 54.4 0 45.6 12.01,2-propanediol 0 4.4 100 0 0 34.1 5 4.8 98.6 1.4 0 32.4 20 3.9 97.6 2.40 19.5 50 4.2 98.5 1.5 0 21.2 ^(a)optical density ^(b)3-hydroxybutyrateunits ^(c)3-hydroxyvalerate units ^(d)3-hydroxypropionate units^(e)percent of dry cell weight

Example 6 Production of poly(3-hydroxypropionate) from Central MetabolicIntermediates

Examples 1-5 demonstrate that it is possible to obtainpoly(3-hydroxypropionate) from glycerol, and, as discussed above, it ispossible in both transgenic and nontransgenic organisms to produceglycerol from central metabolic intermediates. Therefore, a combinationof the two pathways will allow the synthesis ofpoly(3-hydroxypropionate) from central metabolic intermediates. Thesepathways can be combined either by introducing thepoly(3-hydroxypropionate) synthesis genes into a glycerol-producing hostor by introducing glycerol synthesis genes into a host already capableof poly(3-hydroxypropionate) synthesis from glycerol, such as describedin the above examples.

In the former case, genes encoding a vicinal diol dehydratase, a PHAsynthase, and optionally an aldehyde dehydrogenase, 1,3-propanedioloxidoreductase, and hydroxyacyl-CoA transferase are expressed in a hostcapable of producing glycerol from central metabolic intermediates. Anexample of such a host is an Escherichia coli that expresses theSaccharomyces cerevisiae DAR1 (dihydroxyacetone phosphate dehydrogenase)and GPP2 (sn-glycerol-3-phosphate phosphatase) genes (Anton, D.“Biological production of 1,3-propanediol”, presented at UnitedEngineering Foundation Metabolic Engineering II conference, Elmau,Germany, Oct. 27, 1998; PCT WO 98/21339), as described above. Manystrains of E. coli naturally express 1,3-propanediol oxidoreductase andaldehyde dehydrogenase enzymatic activities, but their levels mayoptionally be augmented by mutagenesis or purposeful overexpression ofenzymes that carry out these functions. The additional genes necessarycan be introduced on a plasmid such as pFS48B, which contains, under thecontrol of the trc promoter, 4-hydroxybutyryl-CoA transferase fromClostridium kluyveri; PHA synthase from Zoogloea ramigera and glyceroldehydratase and 1,3-propanediol oxidoreductase from Klebsiellapneumoniae. Any or all of these genes may also be introduced byintegration into the chromosome using standard techniques well-known tothose skilled in the art.

Similarly, the DAR1 and GPP2 genes can be introduced into a host alreadycapable of poly(3-hydroxypropionate) synthesis, such as MBX820/pFS47A,described above. The DAR1 and GPP2 genes may be introduced on a plasmidcompatible with pFS47A (a plasmid that can be maintained simultaneouslywith pFS47A), or they may be integrated into the chromosome. MBX820stably expresses acetoacetyl-CoA thiolase, 3-hydroxybutyryl-CoAreductase, and PHA synthase, and therefore it is capable of synthesizingpoly(3-hydroxybutyrate-co-3-hydroxypropionate). If the homopolymerpoly(3-hydroxypropionate) is desired, a strain expressing only PHAsynthase rather than all three PHB biosynthetic genes may be used.

In order to demonstrate the pathway for the biosynthesis of PHP formglucose, plasmid pMS15 (shown schematically in FIG. 6) was constructedto express the following genes as an operon from the trc promoter: PHBsynthase from A. eutrophus, the 4-hydroxybutyryl-CoA transferase from C.kluyveri, the glycerol dehydratase from Klebsiella, the DAR1 gene fromS. cerevisae, the GPP2 gene from S. cerevisae and the 1,3-propanedioloxidoreductase from K. pneumoniae.

The plasmid pFS51 was constructed by ligating DAR1 and GPP2 PCR productsone at a time to pTrcN. The DAR1 gene was amplified by PCR from S.cerevisiae genomic DNA using the following oligonucleotide primers:

(SEQ ID NO: 3) 5′- CTTCCGGATCCATTCAGGAGGTTTTTATGTCTGCTGCTGCTGATAGA-3′(S. cer. DAR1 5′ BamHI) (SEQ ID NO: 4) 5′-CTTCCGCGGCCGCCTAATCTTCATGTAGATCTAATTC-3′ (S. cer. DAR1 3′ NotI)The GPP2 gene was amplified in the same way using the followingoligonucleotide primers:

(SEQ ID NO: 5) 5′-CTTCCGCGGCCGCATTCAGGAGGTTTTTATGGGATTGACTACTAAACCTC-3′(S. cer. GPP2 5′ NotI) (SEQ ID NO: 6) 5′-CCTTCTCGAGTTACCATTTCAACAGATCGTCC-3′ (S. cer. GPP2 3′ XhoI)

PCR for each gene was carried out with Pfu DNA polymerase (Stratagene;La Jolla, Calif.) in a reaction volume of 50 μL, which contained: 10units Pfu polymerase, 1× reaction buffer provided by the manufacturer,50 pmol of each primer, about 200 ng S. cerevisiae genomic DNA, and 200μM of each dNTP. The thermal profile of the reactions was as follows: 27cycles of (94° C., 1 min; 55° C., 2 min; 72° C., 3 min), then 7 min at72° C. The Pfu polymerase was not added until the reaction mixture hadreached 94° C.

The PCR products were purified from a 1% low-melt agarose gel anddigested with the restriction enzymes whose corresponding sites had beenincluded at the 5′ ends of the primers (BamHI and NotI for DAR1, NotIand Xho1 for GPP2). The vector pTrcN is a version of pTrc99a (Pharmacia;Uppsala, Sweden) modified such that it lacks an NcoI site. pTrcN was cutwith BamHI, NotI, and calf intestinal alkaline phosphatase (CIAP;Promega; Madison, Wis.) for insertion of DAR1 and with NotI, XhoI, andCIAP for insertion of GPP2. The ligations were carried out with T4 DNAligase (New England Biolabs; Beverly, Mass.) according to theinstructions provided by the manufacturer. The products of the ligationswere pFS49 (DAR1) and pFS50 (GPP2). To assemble both genes on oneplasmid, pFS49 was cut with MluI and NotI and the 2.3-kb fragmentcontaining the trc promoter and DAR1 was ligated to pFS50 that had beendigested with the same two enzymes and CIAP. The resulting plasmid,which contained the operon DAR1-GPP2 under control of the trc promoter,was denoted pFS51.

E. coli strain MBX184 containing the plasmid pMS15 (shown schematicallyin FIG. 6) was grown overnight in a 200-mL square bottle at 37° C. in 50mL of LB medium which also contained 100 μg/mL ampicillin. The cellswere removed from this culture by centrifugation for 10 minutes at2000×g, and the cells were resuspended in 50 mL of a glucose medium andincubated for 72 hours at 30° C. with shaking at 200 rpm. The glucosemedium contained, per liter: 6.25 g LB powder; 100 μg ampicillin; 20 gglucose; 50 mmol potassium phosphate, pH 7; 10 μmolisopropyl-β-D-thiogalactopyranoside (IPTG); and 0, 10, or 100 nmolcoenzyme B-12. After the incubation, the cells were removed from themedium by centrifugation for 10 minutes at 2000×g, washed once withwater and centrifuged again, then lyophilized. Gas chromatographic (GC)analysis of the lyophilized cell mass showed that, in the experimentwith 10 nM coenzyme B-12, poly(3HP) made up 0.11% of the dry cellweight; in the experiment with 100 nM coenzyme B-12, poly(3HP) made up0.13% of the dry cell weight; and in the experiment with no coenzymeB-12, poly(3HP) was not detected. No polymer constituents other than 3HPwere found in any case. GC analysis was conducted as follows: 15 to 20mg of lyophilized cell mass was subjected to simultaneous extraction andpropanolysis at 100° C. for 3 hours in 2 mL of a mixture containing (byvolume) 50% 1,2-dichloroethane, 40% 1-propanol, and 10% concentratedhydrochloric acid, with 2 mg/mL benzoic acid added as an internalstandard. The water-soluble components of the resulting mixture wereremoved by extraction with 3 mL water. The organic phase (1 μL at asplit ratio of 1:50 at an overall flow rate of 2 mL/min) was analyzed onan SPB-1 fused silica capillary GC column (30 m; 0.32 mm ID; 0.25 μmfilm; Supelco; Bellefonte, Pa.) with the following temperature profile:80° C. for 2 min; 10 C.° per min to 250° C.; 250° C. for 2 min. Thestandard used to test for the presence of 3HP residues wasβ-propiolactone. Both poly(3HP) and β-propiolactone yield the 1-propylester of 3-hydroxypropionate when subjected to propanolysis.

Example 7 PHBV from Central Metabolic Intermediates

As demonstrated above, it is possible to obtain PHBV from1,2-propanediol with the optional addition of other carbon sources suchas glucose, and it is possible in both transgenic and nontransgenicorganisms to produce 1,2-propanediol from central metabolicintermediates (Cameron et. al., 1998, Biotechnol. Prog. 14 116-125).Therefore, a combination of the two pathways will allow the synthesis ofPHBV from central metabolic intermediates. These pathways can becombined either by introducing the PHBV synthesis genes into a1,2-propanediol-producing host or by introducing 1,2-propanediolsynthesis genes into a host already capable of PHBV synthesis from1,2-propanediol, such as described in the above examples.

In the former case, genes encoding a vicinal diol dehydratase, a PHAsynthase, a 3-ketoacyl-CoA thiolase and reductase, and optionally analdehyde dehydrogenase, 1-propanol oxidoreductase, and hydroxyacyl-CoAtransferase are expressed in a host capable of producing 1,2-propanediolfrom central metabolic intermediates. An example of such a host is anEscherichia coli that expresses rat lens aldose reductase oroverexpresses E. coli glycerol dehydrogenase, as described above. Manystrains of E. coli naturally express 1-propanol oxidoreductase, aldehydedehydrogenase, and propionyl-CoA transferase enzymatic activities, buttheir levels may optionally be augmented by mutagenesis or purposefuloverexpression of enzymes that carry out these functions. The additionalgenes necessary can be introduced as plasmid-borne genes or may beintegrated into the chromosome, or a combination of the two approachesmay be used. For example, a plasmid such as pFS48B, which contains,under the control of the trc promoter, 4-hydroxybutyryl-CoA transferasefrom Clostridium kluyveri; PHA synthase from Zoogloea ramigera andglycerol dehydratase and 1,3-propanediol oxidoreductase from Klebsiellapneumoniae, may be used in combination with integration of the PHBsynthesis genes into the chromosome using standard techniques well-knownto those skilled in the art.

Similarly, the rat lens aldose reductase or E. coli glyceroldehydrogenase genes can be introduced into a host already capable ofPHBV synthesis, such as MBX769/pFS44C, described above. An additionalimprovement may result from the overexpression of a methylglyoxalsynthase gene, as suggested by Cameron et al., 1998 (Biotechnol. Prog.14 116-125). The rat lens aldose reductase or E. coli glyceroldehydrogenase gene may be introduced on a plasmid compatible with pFS44C(a plasmid that can be maintained simultaneously with pFS44C), or theymay be integrated into the chromosome.

Example 8 Identification of 3-hydroxypropionaldehyde DehydrogenaseActivity

The aldH gene sequence from E. coli is available from GENBANK. This genewas cloned into the Acc65I and NotI sites of the cloning vector pSE380following PCR amplification using the approach described in Example 6and the following primers:

ald-Acc65I (SEQ ID NO: 7)5′-ggtggtaccttaagaggaggtttttatgaattttcatcacctggctt ald-NotI(SEQ ID NO: 8) 5′-ggtgcggccgctcaggcctccaggcttatcca

The resulting recombinant plasmid pALDH was introduced into E. coli DH5alpha and grown in 5 ml LB medium with 100 μg/ml ampicillin 37° C. Thenext day a 100 ml containing 100 μg/ml ampicillin was innoculated with100 μl of the overnight culture and grown until the absorbance at 600 nmreached 0.5 at which time the trc promoter was induced with 1 mM IPTGand incubated a further 3 hours at 37° C. The cells were harvested,washed and resuspended in 0.1 M Tris.HCl pH 8.0 and lysed by sonication.The cell lysate was assayed for aldehyde dehydrogenase activity using3-hydroxypropionaldehyde with both NAD and NADP as cofactor. Assays wereperformed using an Hewlett Packard diode array spectrophotometer. Enzymereactions were carried out in 1.5 ml UV cuvettes in a solutioncontaining the following: 0.1 M Tris.Hcl, pH 8.0, 1 mM NAD or NADP, 6 mMdithiothreitol and crude cell extract to a final volume of 1 ml. Themixture was incubated for 20 seconds before initiating the reaction byadding 1 mM 3-hydroxypropionaldehyde and monitoring the reaction at 340nm. The lysate showed significant 3-hydroxypropionaldehyde dehydrogenaseactivity when NAD was the cofactor (1.35 μmoles/min/mg protein) whichwas not present in the control sample prepared using the vector alone.Therefore the aldH gene can be used to increase the3-hydroxypropionaldehyde dehydrogenase activity in the strains describedin the previous examples.

Modifications and variations of the methods and materials describedherein will be obvious to those skilled in the art and are intended tocome within the scope of the following claims.

We claim:
 1. A method for producing 3-hydroxypropionic acid comprisingproviding organisms selected from the group consisting of microorganismsand plants, which are genetically engineered to express one or moreenzymes selected from the group consisting of vicinal diol dehydratase,aldehyde dehydrogenase, 1,3-propanediol oxidoreductase,glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase effectiveto produce 3-hydroxypropionate, providing a polyol substrate which canbe converted into 3-hydroxypropionate by enzymes expressed by theorganisms, culturing the organisms under conditions wherein3-hydroxypropionate accumulates in the organism.
 2. The method of claim1 wherein the organisms are bacteria.
 3. The method of claim 1 whereinthe organisms are plants.
 4. The method of claim 1 wherein the organismsare genetically engineered with plasmids encoding one or more of theenzymes.
 5. The method of claim 1 wherein the organisms are geneticallyengineered to incorporate the genes encoding the enzymes into thechromosome.
 6. The method of claim 1 wherein the substrates are selectedfrom the group consisting of 1,2-propanediol, 1,3 propanediol,glycerol-3-phosphate, and glycerol.
 7. The method of claim 1 wherein thevicinal diol dehydratase is selected from the group consisting ofglycerol dehydratase and diol dehydratase.
 8. The method of claim 1further comprising a substrate selected from the group consisting ofsugars and metabolic intermediates of sugar metabolism.
 9. The method ofclaim 1 wherein the organisms express an aldehyde dehydrogenase and avicinal diol dehydratase.
 10. A system for making 3-hydroxypropionicacid comprising organisms selected from the group consisting ofmicroorganisms and plants genetically engineered to express one or moreenzymes selected from the group consisting of a vicinal dioldehydratase, aldehyde dehydrogenase, 1,3-propanediol oxidoreductase,glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase, whereinthe organisms can convert substrates into 3-hydroxypropionate, and aneffective amount of a polyol substrate to be converted into3-hydroxypropionate under conditions wherein 3-hydroxypropionateaccumulates.
 11. The system of claim 10 wherein the organisms arebacteria.
 12. The system of claim 10 wherein the organisms are plants.13. The system of claim 10 wherein the organisms are geneticallyengineered with plasmids encoding one or more of the enzymes.
 14. Thesystem of claim 10 wherein the organisms are genetically engineered toincorporate the genes encoding the enzymes into the chromosome.
 15. Thesystem of claim 10 further comprising coenzyme B-12.
 16. The system ofclaim 10 wherein the vicinal diol dehydratase is selected from the groupconsisting of glycerol dehydratase and dial dehydratase.
 17. The systemof claim 10 wherein the organisms express an aldehyde dehydrogenase anda vicinal dial dehydratase.
 18. A method of producing3-hydroxypropionyl-CoA from propionic acid according to claim 1comprising providing the organisms with an enzyme or enzymes having theactivity of a 3-hydroxypropionyl-CoA synthetase or3-hydroxypropionyl-CoA transferase which can convert the3-hydroxypropionic acid to 3-hydroxypropionyl-CoA.
 19. A method ofproducing a polyhydroxyalkanoate from 3-hydroxypropionyl-CoA accordingto the method of claim 18 comprising providing the organisms with anenzyme having the activity of a polyhydroxyalkanoate synthase, whereinthe 3-hydroxypropionyl CoA is incorporated into a polyhydroxyalkanoateby the polyhydroxyalkanoate synthase.
 20. The method of claim 19 whereinthe polyhydroxyalkanoate is a 3-hydroxypropionate polymer or copolymer.21. The method of claim 1 comprising providing as a substrate,glycerol-3-phosphate.
 22. The method of claim 1 wherein the organismshave been genetically engineered to express enzymes selected from thegroup consisting of glycerol-3-phosphate dehydrogenase andglycerol-3-phosphatase, which convert the metabolic intermediate toglycerol, which is then converted to 3-hydroxypropionic acid.
 23. Themethod of claim 1 wherein the organisms are further geneticallyengineered to express one or more enzymes selected from the groupconsisting of acyl-CoA transferase, acyl-CoA synthetase, andpolyhydroxyalkanoate synthase.
 24. The system of claim 10 wherein theorganisms are further genetically engineered to express one or moreenzymes selected from the group consisting of acyl-CoA transferase,acyl-CoA synthetase, and polyhydroxyalkanoate synthase.